Methods for modulating mannose content of recombinant proteins

ABSTRACT

The present invention relates to methods of modulating (e.g., reducing) the mannose content, particularly high-mannose content of recombinant glycoproteins.

BACKGROUND OF THE INVENTION

Higher eukaryotes perform a variety of post-translational modifications,including methylation, sulfation, phosphorylation, lipid addition andglycosylation. Such modifications may be of critical importance to thefunction of a protein. Secreted proteins, membrane proteins, andproteins targeted to vesicles or certain intracellular organelles arelikely to be glycosylated.

N-linked glycosylation is a form of glycosylation involving addition ofoligosaccharides to an asparagine residue found in recognition sequences(e.g., Asn-X-Ser/Thr) in proteins. N-linked glycoproteins containstandard branched structures, which are composed of mannose (Man),galactose, N-acetylglucosamine (GlcNAc) and neuramic acids. ProteinN-glycosylation typically originates in the endoplasmic reticulum (ER),where an N-linked oligosaccharide (e.g., Glc₃ Man₉ GlcNAc₂) assembled ondolichol (a lipid carrier intermediate) is transferred to theappropriate Asparagine (Asn) of a nascent protein. This is an eventcommon to all eukaryotic N-linked glycoproteins. There are two majortypes of N-linked saccharides: high-mannose oligosaccharides, andcomplex oligosaccharides.

High-mannose oligosaccharides typically include two N-acetylglucosamineswith many mannose residues (e.g., greater than 4). Complexoligosaccharides are so named because they can contain almost any numberof the other types of saccharides, including more than the original twoN-acetylglucosamines. Proteins can be glycosylated by both types ofoligosaccharides on different portions of the protein. Whether anoligosaccharide is high-mannose or complex is thought to depend on itsaccessibility to saccharide-modifying proteins in the Golgi apparatus.If the saccharide is relatively inaccessible, it will most likely stayin its original high-mannose form. If it is accessible, then it islikely that many of the mannose residues will be cleaved off and thesaccharide will be further modified by the addition of other types ofgroup as discussed above.

After an oligosaccharide chain has been added to a protein, the threeglucose and one mannose residues are removed by three different enzymesin a fixed order. This event occurs in the ER and is a signal that theprotein can be transported to the Golgi for further processing. Afterthe processing in the ER, the high-mannose type oligosaccharide isformed. The three glucose residues and one specific alpha-1,2-linkedmannose residue are removed by specific glucosidases and analpha-1,2-mannosidase in the ER, resulting in the core oligosaccharidestructure, Man₈ GlcNAc₂. The protein with this core sugar structure istransported to the Golgi apparatus where the sugar moiety undergoesvarious modifications.

In mammalian cells, the modification of the sugar chain proceeds via 3different pathways depending on the protein moiety to which it is added.The three different pathways are: (1) the core sugar chain does notchange; (2) the core sugar chain is changed by adding theN-acetylglucosamine-l-phosphate moiety (GlcNAc-l-P) in UDP-N-acetylglucosamine (UDP-GlcNAc) to the 6-position of mannose in the core sugarchain, followed by removing the GlcNAc moiety to form an acidic sugarchain in the glycoprotein; or (3) the core sugar chain is firstconverted into Man₅ GlcNAc₂ by removing 3 mannose residues withmannosidase I; Man₅ GlcNAc₂ is further modified by adding GlcNAc andremoving 2 more mannose residues, followed by sequentially addingGlcNAc, galactose (Gal), and N-acetylneuraminic acid (also called sialicacid (NeuNAc)) to form various hybrid or complex sugar chains (R.Kornfeld and S. Kornfeld, Ann. Rev. Biochem. 54: 631-664 (1985); Chibaet al., J. Biol. Chem. 273: 26298-26304 (1998)).

The oligosaccharide content of recombinant proteins can affect thesafety and efficacy of therapeutic glycoproteins. Accordingly, methodsfor controlling the oligosaccharide content, particularly the mannosecontent, of such glycoproteins would be beneficial.

The high mannose content of glycoprotein compositions, particularlytherapeutic antibodies, can significantly affect the safety and efficacyof such proteins during therapeutic use. Without being bound by aparticular theory, evidence suggests that high-mannose glycoproteins arecleared from circulation faster than their low mannose counterparts dueto, for example, mannose receptors on macrophages and dendritic cells.Additionally, high mannose glycoproteins are expected to be moreimmunogenic. Accordingly, it is desirable to produce therapeuticglycoproteins such as, for example, therapeutic antibodies, having lowmannose content.

The present inventors solves this need in the art by providing methodsfor modulating (e.g., controlling or reducing) the mannose content ofrecombinantly produced proteins and peptides.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery offactors that affect mannose content and, in particular, high-mannosecontent, of recombinantly expressed glycoproteins.

Accordingly, in one aspect, the present invention provides a method ofmodulating the mannose content (i.e., on an oligosaccharide side chain)of a recombinant glycoprotein produced in a mammalian host cell bymanipulating the cell culture conditions such that the glycoproteinproduced by the cell has low-mannose content. As used herein, the term“low-mannose content” refers to glycoprotein compositions wherein lessthan about 10%, or less than about 8%, or less than about 5% (e.g.,about 4% or less) of the glycoproteins in the composition have more than4 mannose residues (i.e., are species of M5 or greater). As used herein,the term “low-mannose content” also refers to glycoprotein compositionswherein less than about 10%, less than about 9%, less than about 8%,less than about 7%, less than about 6%, less than about 5%, less thanabout 4%, less than about 3%, less than about 2%, less than about 1%, orany values between any of these preceding ranges, or even at zero.

In one embodiment of the invention, low-mannose content is achieved bymaintaining the cell culture environment at low osmolality (e.g., lessthan about 600 mOsm/Kg, or less than about 500 mOsm/Kg, or less thanabout 400 mOsm/Kg, e.g., between about 380 to 250 mOsm/Kg). Thisenriches the cell culture for glycoproteins having low mannose-contenti.e., having 4 or fewer mannose residues on the oligosaccharide sidechains of the glycoprotein. Accordingly, in a particular embodiment, theinvention provides a method for producing a recombinant glycoproteinhaving low-mannose content comprising culturing a mammalian host-cell(e.g., in an expansion or production phase of the culture) whichexpresses the glycoprotein in a medium having an osmolality of about 600mOsm/Kg or less (e.g., between a range of about 200 and 600 mOsm/Kg,e.g., about 250 and 550 mOsm/Kg, about 250 and 500 mOsm/Kg, about 250and 450 mOsm/Kg, about 250 and 400 mOsm/Kg, about 250 and 380 mOsm/Kg,or about 250 and 350 mOsm/Kg).

The foregoing osmolality ranges can be achieved by manipulating a numberof cell culture parameters including, but not limited to, concentrationsof one or more of salts, vitamins, sugars, peptones and amino acids inthe cell culture medium. Accordingly, in a particular embodiment, theinvention provides a method of producing a recombinant glycoproteinhaving low-mannose content by culturing a host-cell which expresses theglycoprotein in a medium containing potassium at a concentration ofabout 70 mM or less (e.g., about 10 mM to about 50 mM); and/or sodium ata concentration of about 200 mM or less (e.g., about 50 mM to about 100mM) and maintaining the osmolality of the cell culture at about 600mOsm/Kg or less.

In still another embodiment, the invention provides a method ofproducing a recombinant glycoprotein having low-mannose content byculturing a host-cell which expresses the glycoprotein in a medium whichis substantially free of one or more amino acids selected from the groupconsisting of alanine, arginine, aspartic acid and glutamic acid, andmaintaining the osmolality of the cell culture at about 600 mOsm/Kg orless.

In addition, in still another embodiment, the medium can include one ormore vitamins selected from the group consisting of biotin, D-calciumpantothenate, choline chloride, folic acid, i-inositol, niacinamide,pyridoxal HCl, pyridoxine HCl, riboflavin, thamine HCl andcyanocobalamin, at a concentration of about 0.00005 g/L to about 0.9g/L. In yet another embodiment, the medium includes glucose at aconcentration of about 1 mM to about 90 mM. In a further embodiment, themedium includes one or more peptones selected from the group consistingof yeast extract, yeast hydrolysate, soy peptone, soy hydrolysate, wheatpeptone and wheat hydrolysate, at a concentration of about 0.5 g/L toabout 60 g/L.

In yet a further embodiment of the present invention, the cell culturemedium can include one or more osmoprotectants in an amount necessary tomaintain the osmolality at a desired level, e.g., about 600 mOsm/Kg orless. Suitable osmoprotectants are known in the art and include, forexample, betaine, glycine, L-threonine and L-proline, and derivativesthereof such as, for example, glycine betaine and betaine aldehyde. In aparticular embodiment, the osmoprotectant (e.g., betaine) is present ata concentration of about 20 mM or greater in the cell culture medium. Inparticular embodiments, the osmoprotectant (e.g., betaine) is present ata concentration of about 1 mM to about 100 mM or at about 20 mM to about30 mM.

Additional cell culture parameters that may be controlled, either aloneor in combination with one or more of the parameters described hereininclude, for example, temperature and duration of time which the cellsare cultured for. In certain embodiments, a host-cell expressing arecombinant glycoprotein is cultured at a temperature of about 31° C. toabout 38° C. In certain other embodiments, a host cell expressing arecombinant glycoprotein is cultured for a period ranging from about 5days to about 14 days.

Suitable host cells for expressing recombinant glycoproteins accordingto the present invention are well known in the art and include any ofthose described herein, such as CHO cells, lymphocytic cells (e.g., NSOcells) and a variety of other mammalian cells.

The present invention can be employed to product a wide variety ofglycoproteins having low-mannose content as described herein. In aparticular embodiment, the invention is used to produce a recombinantmonoclonal antibody or an antigen-binding fragment thereof havinglow-mannose content. Suitable antibodies can include, for example,murine, chimeric, humanized and fully human antibodies, as well as otherantibody forms known in the art. In another particular embodiment, theantibody binds IL-15, which includes but are not limited to theantibodies disclosed in U.S. Publication No.: 2003-0138421, which isincorporated by reference herein in its entirety. In another particularembodiment, the antibody is a fully human monoclonal antibody that bindsIL-15 having a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO:4 and/or a heavy chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO:2, as well ashomologous sequences which bind IL-15 (e.g., having amino acid sequencesof about 80, 85, 90, 95% or greater identity to SEQ ID NO: 4 or SEQ IDNO: 2, respectively). In a further particular embodiment, the antibodyis a human antibody that binds IL-15, or an antigen-binding fragmentthereof, having a light chain variable region comprising one or morecomplementarity determining regions (CDRs) set forth in SEQ ID NOs:8-10,as well as homologous sequences which bind IL-15 (e.g., having aminoacid sequences of about 80, 85, 90, 95% or greater identity to any ofSEQ ID NOS: 8-10, respectively), and a heavy chain variable regioncomprising one or more complementarity determining regions (CDRs) setforth in SEQ ID NOs:5-7 as well as homologous sequences which bind IL-15(e.g., having amino acid sequences of about 80, 85, 90, 95% or greateridentity to any of SEQ ID NOS: 5-7, respectively). In a particularembodiment, a human monoclonal antibody that binds IL-15 or anantigen-binding fragment thereof, includes a light chain variable regioncomprising all three CDRs set forth in SEQ ID NOs:8-10, and a heavychain variable region comprising all three CDRs set forth in SEQ ID NOs:5-7, or conservative amino acid substitutions thereof.

In yet another aspect, the present invention provides recombinantglycoproteins having low-mannose content produced by the methodsdescribed herein. Accordingly, such glycoproteins may include any of theaforementioned therapeutic glycoproteins, such as antibodies, hormones,enzymes, peptides and other glycoproteins.

Also encompassed by the present invention are compositions comprisingany of the aforementioned glycoproteins having low-mannose content. In aparticular embodiment, the composition is a pharmaceutical compositionthat includes an isolated glycoprotein (e.g., an isolated humanmonoclonal antibody that binds IL-15 or an antigen binding fragmentthereof) having low-mannose content and a pharmaceutically acceptablecarrier.

Accordingly, in still another aspect, the present invention provides amethod of treating or preventing a disorder that is associated with anoverexpression of human IL-15 and/or in which a downregulation orinhibition of human IL-15 induced effects is beneficial is provided, byadministering to a subject an isolated IL-15 antibody having low-mannosecontent. Exemplary disorders include, but are not limited to,vasculiitis, psoriasis, multiple sclerosis, rheumatoid arthritis,inflammatory bowel disease (e.g., Crohn's disease or celiac disease),allograft rejection, graft versus host disease, T-cell lymphoma, andT-cell leukemia.

Accordingly, in still another aspect, the present invention provides amethod of treating or preventing a disorder that is associated with anoverexpression of human IL-15 and/or in which a downregulation orinhibition of human IL-15 induced effects is beneficial is provided, byadministering to a subject an isolated IL-15 antibody having low-mannosecontent. Exemplary disorders include, but are not limited to,arthritides, connective tissue disorders, ophthalmological disorders,neurological disorders, gastrointestinal and hepatic disorders, allergicdisorders, hematologic disorders, skin disorders, pulmonary disorders,malignancies, transplantation-derived disorders, endocrinologicdisorders, vascular disorders, gynecological disorders and infectiousdiseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the correlation between osmolality andhigh-mannose content of a fully human monoclonal antibody that bindsIL-15 produced by culturing cells expressing the antibody in shakercontrol (50 mL) and bioreactors (150 L and 500 L).

FIG. 2 is a graph depicting the correlation between addition of anosmoprotectant, betaine, and high mannose content of a fully humanmonoclonal antibody that binds IL-15.

FIG. 3 is a graph depicting the correlation between osmolality and K+concentration of culture medium.

FIG. 4 is a graph depicting the correlation between high-mannose contentof a fully human monoclonal antibody that binds IL-15 and osmolality, byculturing cells in a medium containing either 15 mM or 45 mM KCl.

FIG. 5 is a graphical representation of the correlation between the K+concentration and high-mannose content, showing that the optimalconcentration of K+ for keeping the high-mannose content below 10% isbetween about 0 and about 70 mM.

FIG. 6 is a graph representing the correlation between Na+ concentrationand high-mannose content, showing that the optimal concentration of Na+for keeping the high-mannose content below 10% is between about 0 mM andabout 200 mM.

FIG. 7 is a graph depicting the correlation between amino acidconcentration and high-mannose content.

FIG. 8 is a graph depicting the correlation between the type of feedmedium used and high-mannose content.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, it is desirable to produce therapeutic glycoproteins suchas, for example, therapeutic antibodies, having low-mannose content.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

I. DEFINITIONS

Carbohydrate moieties are described herein with reference to commonlyused nomenclature for oligosaccharides. A review of carbohydratechemistry which uses this nomenclature can be found, for example, inHubbard and Ivatt, Ann. Rev. Biochem. 50:555-583 (1981). Thisnomenclature includes, for instance, Man, which represents mannose;GlcNAc, which represents 2-N-acetylglucosamine; Gal which representsgalactose; and Glc, which represents glucose. Sialic acids are describedwith reference to the shorthand notation NeuNAc, for5-N-acetylneuraminic acid, and NeuNGc for 5-glycolylneuraminic acid.

The term “osmolality,” as used herein, refers to a measure of theosmotic pressure of dissolved solute particles in an aqueous solution.The solute particles include both ions and non-ionized molecules.Osmolality is expressed as the concentration of osmotically activeparticles (i.e., osmoles) dissolved in 1 kg of solution (1 mOsm/kg H₂Oat 38° C. is equivalent to an osmotic pressure of 19 mm Hg). As usedherein, the abbreviation “mOsm” means “milliosmoles/kg solution.” Inexemplary embodiments, osmolality of the cell culture medium ismaintained at about 600 mOsm/Kg or less, or at about 550 mOsm/Kg orless, or at about 500 mOsm/Kg or less, or at about 450 mOsm/Kg or less,or at about 400 mOsm/Kg or less, or at about 380 mOsm/Kg or less, orbetween at about 200 mOsm/Kg and about 600 mOsm/Kg, or between at about250 mOsm/Kg and about 550 mOsm/Kg, or between at about 250 mOsm/Kg andabout 500 mOsm/Kg, or between at about 250 mOsm/Kg and about 450mOsm/Kg, or between at about 250 mOsm/Kg and about 400 mOsm/Kg, orbetween at about 250 mOsm/Kg and about 380 mOsm/Kg, or between at about250 mOsm/Kg and about 350 mOsm/Kg.

As used herein, the term “glycoprotein” refers to peptides and proteins,including antibodies, having at least one oligosaccharide side chainincluding mannose residues. Glycoproteins may be homologous to the hostcell, or may be heterologous, i.e., foreign, to the host cell beingutilized, such as, for example, a human glycoprotein produced by aChinese hamster ovary (CHO) host-cell. Such glycoproteins are generallyreferred to as “recombinant glycoproteins.” In certain embodiments,glycoproteins expressed by a host-cell are directly secreted into themedium. Examples of mammalian glycoproteins include the followingmolecules and antibodies against thereto, cytokines, e.g., IL-1 toIL-15, and their receptors; chemokines, such as TNF, TECK, and theirreceptors, e.g., TNFRs, CCR9; growth hormone, including human growthhormone, and bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-l-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; enkephalinase; RANTES(regulated on activation normally T-cell expressed and secreted); humanmacrophage inflammatory protein (MIP-1-alpha); serum albumin such ashuman serum albumin; mullerian-inhibiting substance; relaxin A-chain;relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; amicrobial protein, such as beta-lactamase; DNase; inhibin; activin;vascular endothelial growth factor (VEGF); receptors for hormones orgrowth factors; integrin; protein A or D; rheumatoid factors; aneurotrophic factor such as bone-derived neurotrophic factor (BDNF),neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nervegrowth factor such as NGF-beta; platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, or TGF-beta5;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I), insulin-like growth factor binding proteins; CD proteinssuch as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductivefactors; immunotoxins; bone morphogenetic protein (BMP); interferonssuch as interferon-alpha, -beta, and -gamma; colony stimulating factors(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1to IL-15; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;and regulatory proteins.

As used herein, the terms “cell culture medium” and “culture medium”refer to a nutrient solution used for growing mammalian cells thattypically provides at least one component from one or more of thefollowing categories: 1) an energy source, usually in the form of acarbohydrate such as, for example, glucose; 2) one or more of allessential amino acids, and usually the basic set of twenty amino acidsplus cysteine; 3) vitamins and/or other organic compounds required atlow concentrations; 4) free fatty acids; and 5) trace elements, wheretrace elements are defined as inorganic compounds or naturally occurringelements that are typically required at very low concentrations, usuallyin the micromolar range. The nutrient solution may optionally besupplemented with additional components to optimize growth of cells.

The mammalian cell culture of the present invention is prepared in amedium suitable for the particular cell being cultured. Suitable cellculture media that may be used for culturing a particular cell typewould be apparent to one of ordinary skill in the art. Exemplarycommercially available media include, for example, Ham's F10 (SIGMA),Minimal Essential Medium (MEM, SIGMA), RPMI-1640 (SIGMA), and Dulbecco'sModified Eagle's Medium (DMEM, SIGMA). Any of these or other suitablemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics (such as Gentamycin™), trace elements (defined as inorganiccompounds usually present at final concentrations in the micromolarrange) lipids (such as linoleic or other fatty acids) and their suitablecarriers, and glucose or an equivalent energy source, and/or modified asdescribed herein to facilitate production of recombinant glycoproteinshaving low-mannose content. In a particular embodiment, the cell culturemedium is serum-free.

In certain embodiments, a cell culture medium is optimized so as tomodulate (e.g., reduce) the high-mannose content of a recombinantglycoprotein expressed by a host-cell cultured in such medium. In aparticular embodiment, the mammalian host cell is a CHO cell and asuitable medium contains a basal medium component such as a DMEM/HAMF-12 based formulation with modified concentrations of one or morecomponents such as, for example, amino acids, salts, sugars, peptonesand vitamins, so as to modulate (e.g., reduce) the high-mannose contentof a recombinant glycoprotein expressed by a CHO cell cultured in suchmedium.

The term “growth phase” of a cell culture refers to the period ofexponential cell growth (i.e., the log phase) where the cells aregenerally rapidly dividing. Cells are maintained at the growth phase fora period of about one day, or about two days, or about three days, orabout four days, or longer than four days. The duration of time forwhich the cells are maintained at growth phase will vary based on thecell-type and rate of growth of cells and the culture conditions, forexample.

The term “transition phase” refers to a period of time between thegrowth phase and the production phase. Generally, transition phase isthe time during which culture conditions may be controlled to support ashift from growth phase to production phase. Various cell cultureparameters which may be controlled include but are not limited to, oneor more of, temperature, osmolality, vitamins, amino acids, sugars,peptones, ammonium and salts.

The term “production phase” of a cell culture refers to the period oftime where the cell growth has plateaued. The logarithmic cell growthtypically ends before or during this phase and protein production takesover. It is desirable to supplement the cell culture medium so as toachieve the desired protein production at this stage.

The terms “mammalian host cell,” “host-cell,” and “mammalian cell” referto cell lines derived from mammals that are capable of growth andsurvival when placed in either monolayer culture or in suspensionculture in a medium containing the appropriate nutrients and growthfactors. Typically, such cells are capable of expressing and secretinglarge quantities of a particular glycoprotein of interest into theculture medium. Examples of suitable mammalian host cells include, butare not limited to, Chinese hamster ovary cells/-DHFR (Urlaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); dp12CHO cells (EP307247); monkey kidney CVI line transformed by SV40 (ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture) (Graham et al., J. Gen Virol., 36:59 (1977)); babyhamster kidney cells (ATCC CCL 10); mouse sertoli cells (TM4) (Mather,Bibl. Reprod., 23:243-251 (1980)); monkey kidney cells (ATCC CCL 70);African green monkey kidney cells (VERO-76) (ATCC CRL-1587); humancervical carcinoma cells (HeLa) (ATCC CCL 2); canine kidney cells (MDCK)(ATCC CCL 34); buffalo rat liver cells (BRL 3A) (ATCC CRL 1442); humanlung cells (W138) (ATCC CCL 75); human liver cells (Hep G2 HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The terms “expression,” “express” and “expresses” generally refer totranscription and translation occurring within a host-cell. The level ofexpression of gene product in a host cell may be determined on the basisof either the amount of corresponding mRNA that is present in the cellor the amount of the protein encoded by the gene. For example, mRNAtranscribed from a product gene can be quantitated by northernhybridization. (Sambrook et al., Molecular Cloning: A Laboratory Manual,pp. 7.3-7.57, Cold Spring Harbor Laboratory Press (1989)). A proteinencoded by a gene can be quantitated either by assaying for thebiological activity of the protein or by employing assays that areindependent of such activity, such as, for example, western blottinganalysis or radioimmunoassay using antibodies that are capable ofreacting with the protein. (Sambrook et al., Molecular Cloning: ALaboratory Manual, pp. 18.1-18.88 Cold Spring Harbor Laboratory Press(1989)). In some embodiments, the terms “expression,” “express” and“expresses” are used in reference to a recombinant protein havinglow-mannose content produced by a method of the invention.

The terms “low-mannose” and “low-mannose content,” as used herein, referto a glycoprotein composition, where no more than about 10% of thecomposition comprises glycoproteins having more than 4 mannose residues,i.e., species M5 or greater. Conversely, “high-mannose content” refersto a glycoprotein composition where more than about 10% of thecomposition comprises glycoproteins having more than 4 mannose residues.The terms “low mannose” and “low mannose content,” are also used inreference to a glycoprotein composition including greater than about90%, or greater than about 95% of the composition having glycoproteinsincluding 4 or fewer than 4 mannose residues.

The term “a glycoprotein having low-mannose content” is used inreference to a recombinant glycoprotein composition, which when producedby culturing a host-cell, includes, but are not limited thereto, no morethan about 4%, no more than about 5%, no more than between about 4% andabout 5%, no more than about 6%, no more than between about 5% and 6%,no more than about 7%, no more than between about 6% and 7%, no morethan about 8%, no more than about 7% and 8%, no more than about 9%, nomore than between 8% and 9%, no more than about 10%, or no more thanbetween about 9% and 10% of the glycoproteins in the composition havinggreater than 4 mannose residues (i.e., species MS or greater).Accordingly, the term “a glycoprotein having low-mannose content” refersto a recombinant glycoprotein composition, which when produced byculturing a host-cell, includes greater than about 90%, or greater thanabout 95%, of the glycoproteins in the composition having 4 or fewerthan 4 mannose residues (i.e., 0-4 mannose residues).

The high-mannose content can be measured by one or more methodswell-known in the art, for instance, as described in Wuhrer et al.(Journal of Chromatography B Vol. 825:124-133, 2005) and Dell et al.(Science Vol. 291:2351-2356), and those described herein including, forexample, the analytical method for N-Glycan mapping of glycoproteinsBriefly, N-glycans are removed enzymatically from the recombinantglycoproteins, such as a recombinant monoclonal antibody, and labeledwith a fluorescent tag (2-Aminobenzamide) at the reducing terminus. Thefluorescent N-glycans are separated by high pH anion exchangechromatography (HPAEC), and detected using fluorescence detection.Separation of the neutral N-glycans is generally based on the increasingcomplexity in the N-glycan structures. Separation of the chargedN-glycans is based on the number and type of sialic acid, sulfate, orother modifications present from which a charge number can be derived.These glycan profiles of test samples are compared visually to anappropriate standard.

The high-mannose content can also be measured using a method instantlydisclosed herein: a high-throughput method for detecting and/orquantitating the high-mannose content of a glycoprotein, including butnot limited to, antibody or fragments thereof, e.g., Fab fragments,fusion proteins comprising Fc fragments and peptibody when expressed ineukaryotic host cells. Antibodies typically have a single N-linkedglycan on the Fc region. Because of the partially buried structure ofthe glycan, it is often only partially processed, resulting in excesshigh mannose and hybrid types. Clone selection, mutation of cells orother genetic manipulation, or cell culture manipulation can alter thetypes of glycans produced by the cells. Large numbers ofconditions/cells are explored thus many glycan tests are required duringscreening. Traditional glycan mapping is slow and labor intensive,requiring multiple days. The high-mannose/hybrid glycan assay of thepresent invention provides ratios of glycan types much faster with muchless operator effort.

In particular, the invention provides a method for detecting and/orquantitating the high-mannose content of a glycoprotein in a sample or acomposition comprising said glycoprotein, said method comprisessubjecting the sample or the composition comprising the glycoprotein toan endoglycosidase digestion, reducing the digested glycoproteins usinga reducing agent (if required), and separating the digestedglycoproteins by denature electrophoresis whereby the ratio ofhigh-mannose/hybrid type glycan is determined by subtracting thefraction of non-glycosylated heavy chain (peak fraction withoutendoglycosidase treatment) from the fraction of de-glycosylated heavychain (peak following endoglycosidase digestion). The non-glycosylatedheavy chain fraction or the peak fraction without endoglycosidasetreatment is generated by subjecting the same sample or composition tothe same digestion condition except that no endoglycosidase is presenttherein. This step can be carried out concurrently with or separatelyfrom the endoglycosidase digestion step.

Any endoglycosidases that selectively cleave high mannose and hybridglycans between GlcNAc1 and GlcNAc2 on the core glycan (or generatingshort glycans on the protein), while leaving complex N-linked glycansintact can be used in this invention. For proper quantitation,endoglycosidase must not be in limiting quantities. The specificcondition for carrying out the endoglycosidase digestion, including theconcentration of the enzyme, the incubation temperature and digestiontime, depends on the type of endoglycosidase used. Examples ofendoglycosidases related to this invention include but are not limitedto Endoglycosidase H and Endoglycosidase F1. In one embodiment of thepresent invention, the sample comprising the glycoproteins is treatedwith Endoglycosidase H at 37° C. for about 2 hours, reduced withβ-mercaptoethanol, and subjected to CE-SDS analysis.

Example methods for separating the de-glycosylated glycoproteins, e.g.,de-glycosylated antibody, from the glycosylated glycoproteins, e.g.,glycosylated antibody, include but are not limited to the following twomethods:

1) CE-SDS under reducing conditions. The glycosylated glycoprotein,e.g., an antibody, is denatured with SDS and a reducing agent and theheavy chain (HC) thereof with the glycan is separated from the cleavedHC (de-glycosylated HC) by Capillary Electrophoresis-SDS (CE-SDS). Anelectropherogram is generated of the UV signal. The areas under thepeaks are proportional to the relative amounts. Therefore the amount ofHigh-mannose/hybrid type is determined from the fraction eluting at theearlier de-glycosylated HC position. Since the GlcNAc-HC co-migrateswith de-glycosylated HC, the % de-glycosylated HC from an undigestedsample is subtracted from pre-peak of a digested sample to yield the %high mannose value. Separation requires 15-30 minutes, depending on theconfiguration.

2) Microfluidic-based CE-SDS. The glycoprotein is denatured as in 1) butseparated using a “lab on a chip” instrument, such as the LC90 byCaliper. The same principle is used in the assay and the separation,though a fluorescent dye is used to detect the protein. Separation timeis reduced to about 30 seconds per assay and it can be sampled from amicrotiter plate.

The method of the present invention as described above can be performedon purified protein but also on crude cell culture samples. Withrecombinant antibodies, the signal is strong enough that purification isnot required.

In certain embodiments, glycoproteins having more than 4 mannoseresidues include glycoproteins having 5 to 9 mannose residues (i.e.,species M5-M9). Without wishing to be bound by a particular theory, oneof ordinary skill in the art will understand that a glycoproteincomposition expressed by a host-cell includes glycoproteins with varyingnumber of mannose residues. For example, the low-mannose glycoproteinshave 4 or fewer than 4 mannose residues (e.g., 0-4 mannose residues),and the high-mannose glycoproteins have greater than 4 mannose residues(e.g., M5 species or higher).

In a particular embodiment of the invention, a glycoprotein havinglow-mannose content is a recombinant antibody or an antigen-bindingfragment thereof. In another particular embodiment of the invention, arecombinant glycoprotein having low-mannose content is a humanmonoclonal antibody that binds IL-15 or an antigen-binding fragmentthereof.

The term “substantially free,” as used herein, generally refers topreparations of a cell culture medium which is free or has a reducedamount (i.e., relative to unmodified culture medium) of certaincomponents. For example, in one embodiment, the culture medium used forproducing recombinant glycoproteins having low mannose content issubstantially free of certain amino acids (e.g., one or more amino acidsselected from the group consisting of alanine, arginine, aspartic acidand glutamic acid). In some embodiments, a culture medium substantiallyfree of one or more components is modified to include less than about1%, or less than about 3%, or less than about 5%, or less than about 10%of one or more such components relative to the unmodified culturemedium.

The terms “IL-15,” “IL-15 antigen” and interleukin 15″ are usedinterchangeably herein, and include any variants or isoforms which arenaturally expressed by cells.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The V_(H) and V_(L) regions canbe further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The terms “antigen-binding portion” and “antigen-binding fragment” of anantibody (or simply “antibody portion”), as used herein, refer to one ormore fragments of an antibody that selectively bind to an antigen (e.g.,IL-15). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of antigen-binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), CL and CH1 domains;(ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., Nature 341:544-546 (1989)),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al.Science 242:423-426 (1988); and Huston et al. Proc. Natl. Acad. Sci. USA85:5879-5883 (1988). Such single chain antibodies are also intended tobe encompassed within the terms “antigen-binding portion” and“antigen-binding fragment” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

The term “monoclonal antibody” as used herein, refers to an antibodywhich displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody which displays a single binding specificity andwhich has variable and constant regions derived from human germlineimmunoglobulin sequences. In one embodiment, human monoclonal antibodiesare produced by a hybridoma which includes a B cell obtained from atransgenic non-human animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies can be subjected to in vitro mutagenesis(or, when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. This termrefers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

An “isolated antibody,” as used herein, refers to an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toIL-15 is substantially free of antibodies that specifically bindantigens other than IL-15). An isolated antibody that specifically bindsto an epitope of IL-15 may, however, have cross-reactivity to otherrelated cytokines or to other IL-15 proteins from different species.However, the antibody preferably always binds to human IL-15. Inaddition, an isolated antibody is typically substantially free of othercellular material and/or chemicals. In a particular embodiment, acombination of “isolated” monoclonal antibodies having different IL-15specificities are combined in a well defined composition.

As used herein, “specific binding,” “selective binding” and “selectivelybinds,” refer to an antibody or a fragment thereof, binding to apredetermined antigen. For example, in one embodiment, the antibodybinds with an affinity (K_(D)) of approximately less than 10⁻⁷ M, suchas approximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower whendetermined by surface plasmon resonance (SPR) technology in a BIACORE3000 instrument using recombinant human IL-15 as the analyte and theantibody as the ligand, and binds to the predetermined antigen with anaffinity that is at least two-fold greater than its affinity for bindingto a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich selectively binds to an antigen.”

The term “K_(D),” as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, “non-switched isotype” refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the CH gene encoding the nonswitched isotype is typically the first CHgene immediately downstream from the functionally rearranged VDJ gene.Isotype switching has been classified as classical or non-classicalisotype switching. Classical isotype switching occurs by recombinationevents which involve at least one switch sequence region in thetransgene. Non-classical isotype switching may occur by, for example,homologous recombination between human σ_(μ) and human Σ_(μ)(δ-associated deletion). Alternative non-classical switching mechanisms,such as intertransgene and/or interchromosomal recombination, amongothers, may occur and effectuate isotype switching.

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a μ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule,” as used herein, refers to DNA and RNAmolecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., V_(H),V_(L), CDR3) that selectively bind to IL-15, refer to a nucleic acidmolecule in which the nucleotide sequences encoding the antibody orantibody portion are free of other nucleotide sequences encodingantibodies or antibody portions that bind antigens other than IL-15,which other sequences may naturally flank the nucleic acid in humangenomic DNA. SEQ ID NOS: 1-4 correspond to the nucleotide and amino acidsequences comprising the heavy chain (V_(H)) and light chain (V_(L))variable regions of a human anti-IL-15 antibody. In particular, SEQ IDNO:1 and 2 correspond to the V_(H) of the antibody and SEQ ID NO:3 and 4correspond to the V_(L) of the antibody.

In a particular embodiment, a human monoclonal antibody that bindsIL-15, or an antigen binding fragment thereof, includes a light chainvariable region comprising one or more and preferably all three CDRs setforth in SEQ ID NOs:8-10 and a heavy chain variable region comprisingone or more and preferably all three CDRs set forth in SEQ ID NOs:5-7.

In a particular embodiment, the present invention also encompasses“conservative sequence modifications” or “conservative sequencesubstitutions” of the sequences set forth in SEQ ID NOs:1-10, i.e.,nucleotide and amino acid sequence modifications which do notsignificantly affect or alter the binding characteristics of theantibody encoded by the nucleotide sequence or containing the amino acidsequence. Such conservative sequence modifications include nucleotideand amino acid substitutions, additions and deletions. Modifications canbe introduced into SEQ ID NOs:1-10 by standard techniques known in theart, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions include ones in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-IL-15 antibodyis preferably replaced with another amino acid residue from the sameside chain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of an anti-IL-15 antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-IL-15antibodies can be screened for binding activity.

Accordingly, antibodies encoded by the (heavy and light chain variableregion) nucleotide sequences disclosed herein and/or containing the(heavy and light chain variable region) amino acid sequences disclosedherein (i.e., SEQ ID NOs: 1-4) include substantially similar antibodiesencoded by or containing similar sequences which have beenconservatively modified. Further, discussion as to how suchsubstantially similar antibodies can be generated based on the partial(i.e., heavy and light chain variable regions) sequences disclosedherein as SEQ ID Nos:1-4 is provided below.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

For amino acid sequences, the term “homology” indicates the degree ofidentity between two amino acid sequences when optimally aligned andcompared with appropriate insertions or deletions.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. J. Mol. Biol. 215:403-10 (1990). BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) can be used. (See http://www.ncbi.nlm.nih.gov).

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “substantially pure” when purified away from othercellular components or other contaminants, e.g., other cellular nucleicacids or proteins, by standard techniques, including alkaline/SDStreatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

Nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof, may be mutated, inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the present invention is intended toinclude such other forms of expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject having an inflammatory disease,such as arthritis, e.g., rheumatoid arthritis. The term “non-humananimal” includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, reptiles,etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. FACTORS EFFECTING MANNOSE CONTENT

(a) Osmolality

Various cell culture parameters can affect the mannose content of arecombinant glycoprotein expressed in mammalian cell culture. Inparticular, it was discovered by way of the present invention that thehigher the osmolality of the cell culture medium is, the higher thepercentage of glycoproteins in the composition having more than 4mannose residues (i.e., M5 species or higher) is. Accordingly, in oneembodiment of the present invention, osmolality of the cell culturemedium is maintained at less than about 600 mOsm/Kg to reduce or controlmannose content of expressed glycoproteins (e.g., about 250 mOsm/Kg toabout 600 mOsm/Kg).

For mammalian cell culture, osmolality of the cell culture medium ismaintained at less than about 550 mOsm/Kg, or at less than about 500mOsm/Kg, or at less than about 450 mOsm/Kg, or at less than about 400mOsm/Kg, or at less than about 380 mOsm/Kg, or at between about 200mOsm/Kg and about 600 mOsm/Kg, or at between about 250 mOsm/Kg and about550 mOsm/Kg, or at between about 250 mOsm/Kg and about 500 mOsm/Kg, orat between about 250 mOsm/Kg and about 450 mOsm/Kg, or at between about250 mOsm/Kg and about 400 mOsm/Kg, or at between about 250 mOsm/Kg andabout 380 mOsm/Kg, or at between about 250 mOsm/Kg and about 350mOsm/Kg.

In order to achieve an osmolality in the desired range, theconcentration of various constituents in the culture medium can beadjusted. For example, solutes which can be added to the culture mediumso as to increase the osmolality thereof include proteins, peptides,amino acids, hydrolyzed animal proteins such as peptones,non-metabolized polymers, vitamins, ions, salts, sugars, metabolites,organic acids, lipids, and the like. It will be appreciated however,that the concentration(s) of other constituents in the culture mediumcan be modified in order to achieve a desired osmolality.

In other embodiments, osmolality can be adjusted to the aforementionedranges by adding one or more osmoprotectants to the culture medium.Exemplary osmoprotectants are well known in the art and include, but arenot limited to, betaine, glycine, L-threonine, L-prolinc and derivativesthereof including, but not limited to, glycine betaine, betainealdehyde. In a particular embodiment, a cell culture medium containsbetaine at a concentration of about 20 mM or greater, or at about 1 mMto about 100 mM, and more preferably at about 20 mM to about 30 mM.

Osmolality can be measured by any of the means that are well-known inthe art and those described herein. For example, an osmometer such assold by Fisher Scientific, Pittsburgh, Pa. under the brandname OSMETTEcan be used for measuring osmolity of a cell culture medium.Alternatively, Osmette model 2007 (Precision Systems, Inc., Natick,Mass.) can be used.

In other embodiments of the invention, osmolality can be adjusted bymodifying the concentration of one or more of salts, sugars, peptones,amino acids and ammonium in the cell culture medium.

In still other embodiments, the aforementioned parameters affectingosmolality can be combined with manipulating the temperature andduration of time which the cells are cultured to modulate (e.g., reduce)mannose-content. Accordingly, it should be understood that the variouscell culturing parameters described herein can be adjusted alone or incombination to modulate the mannose-content of recombinantglycoproteins.

(i) Potassium and Sodium Concentrations

In the experiments leading up to the present invention, it wasdemonstrated that an increase in potassium (K+) concentration in theculture medium contributes to the high-mannose content of glycoproteins.Accordingly, in one embodiment, the invention employs a cell culturemedium having a K+ concentration of about 70 mM or less (e.g., at about10 mM to about 50 mM).

As discussed above, the potassium concentration of the cell culturemedium alone may be controlled or it may be controlled in combinationwith one or more of the other factors described herein which affectosmolality. In a particular embodiment, the culture medium furtherincludes a sodium concentration of about 200 mM or less (e.g., at about50 mM to about 100 mM).

(ii) Amino Acids

Other factors which were discovered to affect osmolality of the cellculture medium and/or contribute to high-mannose content ofrecombinantly expressed proteins are the concentration and type of aminoacids in the medium. For example, in a particular embodiment, a doublingof the concentration of all 20 amino acids in the medium results in anincrease in mannose-content. Accordingly, in a particular embodiment ofthe present invention, the cell culture medium is adjusted to have areduced amino acid concentration. In a particular medium, the amino acidconcentration is reduced by about half.

In another particular embodiment, the cell culture medium issubstantially free of one or more amino acids selected from the groupconsisting of alanine, arginine, aspartic acid and glutamic acid.

(iii) Sugars

Other factors which were discovered to affect osmolality of the cellculture medium and/or contribute to high-mannose content ofrecombinantly expressed proteins are the concentration and type ofsugars in the medium. In a particular embodiment, the cell culturemedium includes glucose at a concentration of about 1 mM to about 90 mM.

(iv) Ammonium

Another factor which can affect osmolality of the cell culture mediumand/or contribute to high-mannose content of recombinantly expressedproteins is the ammonium concentration of about 30 mM or less (e.g., atabout 0 mM to about 10 mM). In one embodiment, the ammoniumconcentration is about 10 mM or less.

(v) Peptones

Other factors which were discovered to affect osmolality of the cellculture medium and/or contribute to high-mannose content ofrecombinantly expressed proteins are the concentration and type ofpeptones used in the medium. Peptones are media supplements that areproduced from hydrolyzed animal proteins. Sources of peptones are wellknown in the art and include, for example, animal by-products, gelatinsand plant materials. Exemplary peptones include, but are not limited to,yeast extract, yeast hydrolysate, soy peptone, soy hydrolysate, wheatpeptone, and wheat hydrolysate, at a concentration of about 0.5 g/L toabout 60 g/L.

(vi) Vitamins

Other factors which were discovered to affect osmolality of the cellculture medium and/or contribute to high-mannose content ofrecombinantly expressed proteins are the concentration and type ofvitamins used in the medium. In a particular embodiment, the cellculture medium includes one or more vitamins selected from the groupconsisting of biotin, D-calcium, pantothenate, choline chloride, folicacid, i-inositol, niacinamide, pyridoxal HCl, pyridoxine HCl,riboflavin, thiamine HCl, cyanocobalamin at a concentration of about0.00005 g/L to about 0.9 g/L.

(b) Temperature

Another factor which was discovered to contribute to high-mannosecontent of recombinantly expressed proteins is the temperature at whichthe cell culture is maintained. Accordingly, in another embodiment ofthe present invention, the temperature at which the host-cells arecultured is also adjusted alone or in combination with the foregoingfactors (e.g., adjustment of cell culture timing and factors affectingosmolality) to modulate (e.g., reduce) mannose-content of recombinantlyexpressed glycoproteins. In certain embodiments, host-cells are culturedat about 31° C., or at about 32° C., or at about 33° C., or at about 34°C., or at about 35° C., or at about 36° C., or at about 37° C. or atabout 38° C.

III. CELL CULTURE PROCEDURES

In accordance with the methods of the present invention, host-cells arecultured in a medium that allows for the expression of recombinantglycoproteins having low-mannose content. Suitable cell cultureprocedures and conditions are well known in the art. Host-cells (e.g.,CHO and NSO cells) may be cultured in a wide variety of formats andculture vessels. For example, host-cells may be cultured in formatsdesigned for large scale or small scale production of glycoproteins.Additionally, host-cells may be cultured adherent to the bottom ofculture flasks or dishes, or they may be in suspension in stirredflasks, bioreactors or in roller bottle cultures. In certainembodiments, for production of recombinant glycoproteins in commerciallyrelevant quantities, host-cells may be grown in bioreactors, andpreferably bioreactors having a capacity of about 2 liters or more, orabout 5 liters or more, or about 10 liters or more, or about 50 litersor more, or about 100 liters or more, or about 500 liters or more, orabout 1000 liters or more, or about 1500 liters or more, or about 2000liters or more.

In certain embodiments, host-cells can be cultured (e.g., maintainedand/or grown) in liquid media and preferably are cultured, eithercontinuously or intermittently, by conventional culturing methods suchas standing culture, test tube culture, shaking culture (e.g., rotaryshaking culture, shake flask culture, etc.), aeration spinner culture,or fermentation. In certain embodiments, host-cells are cultured inshake flasks. In yet other embodiments, host-cells are cultured in afermentor (e.g., in a fermentation process). Fermentation processesinclude, but are not limited to, batch, fed-batch and continuous methodsof fermentation. The terms “batch process” and “batch fermentation”refer to a closed system in which the composition of media, nutrients,supplemental additives and the like is set at the beginning of thefermentation and not subject to alteration during the fermentation;however, attempts may be made to control such factors as pH and oxygenconcentration to prevent excess media acidification and/or microorganismdeath. The terms “fed-batch process” and “fed-batch” fermentation referto a batch fermentation with the exception that one or more substratesor supplements are added (e.g., added in increments or continuously) orthe cell culture conditions are changed as the fermentation progresses.The terms “continuous process” and “continuous fermentation” refer to asystem in which a defined fermentation media is added continuously to afermentor and an equal amount of used or “conditioned” media issimultaneously removed, for example, for recovery of the desired product(e.g., recombinant glycoprotein). A variety of such processes have beendeveloped and are well-known in the art.

In a particular embodiment, a host-cell expressing a recombinant humanmonoclonal antibody that binds IL-15 is grown in roller bottles,two-liter spinner-flasks or another suitable culture system.

IV. RECOVERY OF THE GLYCOPROTEIN

Following the polypeptide production phase, the recombinant glycoproteinof interest can be recovered from the culture medium using techniqueswhich are well established in the art. The glycoprotein of interestpreferably is recovered from the culture medium as a secretedpolypeptide, although it also may be recovered from host cell lysates.

In certain embodiments, the culture medium or lysate is centrifuged toremove particulate cell debris. The glycoprotein thereafter is purifiedfrom contaminant soluble proteins and polypeptides using a suitablepurification procedures. Exemplary purification procedures include, butare not limited to, fractionation on immunoaffinity or ion-exchangecolumns; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; and protein A Sepharose columns to removecontaminants such as IgG. A protease inhibitor such as phenyl methylsulfonyl fluoride (PMSF) also may be useful to inhibit proteolyticdegradation during purification. One skilled in the art will appreciatethat purification methods suitable for the recombinant glycoprotein ofinterest may require modification to account for changes in thecharacter of the glycoprotein upon expression in recombinant cellculture.

In a particular embodiment of the present invention, a recombinantglycoprotein expressed using the methods of the present invention is ahuman monoclonal antibody or an antigen-binding fragment thereof.Generally, the antibodies are initially characterized by ELISA. Forexample, microtiter plates can be coated with purified antigen such as,for example, IL-15 in PBS, and then blocked with irrelevant proteinssuch as bovine serum albumin (BSA) diluted in PBS. Dilutions of extractsfrom cultured cells are added to each well and incubated for 1-2 hoursat 37° C. The plates are washed with PBS/Tween 20 and then incubatedwith a goat-anti-human IgG Fc-specific polyclonal reagent conjugated toalkaline phosphatase for 1 hour at 37° C. After washing, the plates aredeveloped with ABTS substrate, and analyzed at OD of 405.

To determine if the antibodies produced by the methods of the presentinvention bind to unique epitopes, each antibody can be biotinylatedusing commercially available reagents (Pierce, Rockford, Ill.).Biotinylated MAb binding can be detected with a streptavidin labeledprobe. To determine the isotype of purified antibodies, isotype ELISAscan be performed using art recognized techniques. For example, wells ofmicrotiter plates can be coated with 10 μg/ml of anti-human Ig overnightat 4° C. After blocking with 5% BSA, the plates are reacted with 10μg/ml of antibodies or purified isotype controls, at ambient temperaturefor two hours. The wells can then be reacted with either human IgG1 orother human isotype specific conjugated probes. Plates are developed andanalyzed as described above.

In a particular embodiment, a recombinant glycoprotein produced usingthe methods of the present invention is a human monoclonal antibody thatbinds IL-15 or an antigen-binding fragment thereof. To test the bindingof IL-15 monoclonal antibodies to live cells expressing IL-15, flowcytometry can be used. Briefly, cell lines and/or human PBMCs expressingmembrane-bound IL-15 (grown under standard growth conditions) are mixedwith various concentrations of monoclonal antibodies in PBS containing0.1% BSA and 0.01% NaN3 at 4° C. for 1 hour. After washing, the cellsare reacted with Fluorescein-labeled anti-human IgG antibody under thesame conditions as the primary antibody staining. The samples can beanalyzed by FACScan instrument using light and side scatter propertiesto gate on single cells and binding of the labeled antibodies isdetermined. An alternative assay using fluorescence microscopy may beused (in addition to or instead of) the flow cytometry assay. Cells canbe stained exactly as described above and examined by fluorescencemicroscopy. This method allows visualization of individual cells, butmay have diminished sensitivity depending on the density of the antigen.

Anti-IL-15 human IgGs can be further tested for reactivity with theIL-15 antigen by Western blotting. Briefly, cell extracts fromhost-cells expressing IL-15 can be prepared and subjected to sodiumdodecyl sulfate polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens will be transferred tonitrocellulose membranes, blocked with 20% mouse serum, and probed withthe monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

V. PHARMACEUTICAL COMPOSITIONS

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofrecombinant glycoproteins having low-mannose content. In a particularembodiment, the pharmaceutical composition includes at least onetherapeutic protein having low-mannose content such as, for example, atherapeutic antibody or an antigen-binding fragment thereof havinglow-mannose content (e.g., a human monoclonal antibody that binds IL-15or an antigen-binding fragment thereof). In another particularembodiment, a pharmaceutical composition of the present inventionincludes one or more recombinant glycoproteins having low mannosecontent, formulated together with a pharmaceutically acceptable carrier.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one or more additional therapeutic agents, such asanti-inflammatory agents, DMARDs (disease-modifying anti-rheumaticdrugs), immunosuppressive agents, chemotherapeutics, and psoriasisagents. The pharmaceutical compositions of the invention can also beadministered in conjunction with radiation therapy. Co-administrationwith other antibodies, such as CD4 specific antibodies and IL-2 specificantibodies, are also encompassed by the invention. Such combinationswith CD4 specific antibodies or IL-2 specific antibodies are consideredparticularly useful for treating autoimmune diseases and transplantrejections.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the recombinant glycoprotein, e.g., anantibody, bispecific and multispecific molecule, may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al., J.Pharm. Sci. 66:1-19 (1977)). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., J. Neuroimmunol. 7:27(1984)).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, the humanantibodies of the invention may be administered once or twice weekly bysubcutaneous injection or once or twice monthly by subcutaneousinjection.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.001 percent to about ninetypercent of active ingredient, preferably from about 0.005 percent toabout 70 percent, most preferably from about 0.01 percent to about 30percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.001 to 90% (morepreferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acomposition of the invention will be that amount of the compound whichis the lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.It is preferred that administration be intravenous, intramuscular,intraperitoneal, or subcutaneous, preferably administered proximal tothe site of the target. If desired, the effective daily dose of atherapeutic composition may be administered as two, three, four, five,six or more sub-doses administered separately at appropriate intervalsthroughout the day, optionally, in unit dosage forms. While it ispossible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the therapeutic glycoproteins of the presentinvention can be formulated to ensure proper distribution in vivo. Forexample, the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. RanadeJ. Clin. Pharmacol. 29:685 (1989). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., Biochem. Biophys. Res. Commun. 153:1038(1988)); antibodies (P. G. Bloeman et al. FEBS Lett. 357:140 (1995); M.Owais et al. Antimicrob. Agents Chemother. 39:180 (1995)); surfactantprotein A receptor (Briscoe et al. Am. J. Physiol. 1233:134 (1995)),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p120(Schreier et al. J. Biol. Chem. 269:9090 (1994)); see also K. Keinanen;M. L. Laukkanen FEBS Lett. 346:123 (1994); J. J. Killion; I. J. FidlerImmunomethods 4:273 (1994). In one embodiment of the invention, thetherapeutic compounds of the present invention are formulated inliposomes; in a more preferred embodiment, the liposomes include atargeting moiety. In a most preferred embodiment, the therapeuticcompounds in the liposomes are delivered by bolus injection to a siteproximal to the tumor or infection. The composition must be fluid to theextent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi.

In a further embodiment, a recombinant glycoprotein of the presentinvention can be formulated to prevent or reduce the transport acrossthe placenta. This can be done by methods known in the art, e.g., byPEGylation of the antibody or by use of F(ab)2′ fragments. Furtherreferences can be made to “Cunningham-Rundles C, Zhuo Z, Griffith B,Keenan J. (1992) Biological activities of polyethylene-glycolimmunoglobulin conjugates.” Resistance to enzymatic degradation. JImmunol Methods. 152:177-190; and to Landor M. (1995) Maternal-fetaltransfer of immunoglobulins, Ann Allergy Asthma Immunol 74:279-283. Thisis particularly relevant when the glycoprotein is an antibody used fortreating or preventing recurrent spontaneous abortion.

A “therapeutically effective dosage” for rheumatoid arthritis preferablywill result in an ACR20 Preliminary Definition of Improvement in thepatients, more preferred in an ACR50 Preliminary Definition ofImprovement and even more preferred in an ARCD70 Preliminary Definitionof Improvement.

ACR20 Preliminary Definition of Improvement is defined as:

≧20% improvement in: Tender Joint Count (TCJ) and Swollen Joint Count(SWJ) and ≧20% improvement in 3 of following 5 assessments: Patient PainAssessment (VAS), Patient Global assessment (VAS), Physician GlobalAssessment (VAS), Patent Self-Assessed Disability (HAQ), Acute PhaseReactant (CRP or ESR).

ACR50 and ACR70 are defined in the same way with ≧50% and ≧70%improvements, respectively. For further details see Felson et al. inAmerican College of Rheumatology Preliminary Definition of Improvementin Rheumatoid Arthritis; Arthritis Rheumatism 38: 727-735 (1995).

The ability of a compound to inhibit cancer can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit, such inhibition invitro by assays known to the skilled practitioner. A therapeuticallyeffective amount of a therapeutic compound can decrease tumor size, orotherwise ameliorate symptoms in a subject. One of ordinary skill in theart would be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

The ability of the antibodies to treat or prevent psoriasis can also beevaluated according to methods well known in the art.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

Other embodiments of the present invention are described in thefollowing Examples, which should not be construed as further limiting.The contents of Sequence Listing, figures and all references, patentsand published patent applications cited throughout this application areexpressly incorporated herein by reference.

EXAMPLES

In all the Examples discussed below, a fully human monoclonal antibodythat binds IL-15 having a light chain variable region comprising theamino acid sequence set forth in SEQ ID NO:4 and a heavy chain variableregion comprising the amino acid sequence set forth in SEQ ID NO:2 wasused as an exemplary recombinant glycoprotein (referred to in theExamples as an “exemplary recombinant glycoprotein”). However, it wouldbe clear to one of ordinary skill in the art that the mannose content ofany recombinant glycoprotein can be modulated, as discussed herein.

Example 1 Osmolality Affects Mannose-Content of RecombinantGlycoproteins

In order to investigate the affect of osmolality on mannose-content ofglycoproteins, mannose-content of an exemplary recombinant glycoproteinwas analyzed at varying osmolalities in both shaker flask and bioreactorcultures. As demonstrated in FIG. 1, high-mannose content increased fromabout 14% to about 24% with the increase of medium osmolality from about500 to about 580 mOsmo/Kg.

In a further experiment, 20 mM of an osmoprotectant, betaine, was addedto the cell culture to provide further evidence regarding therelationship between osmolality and high-mannose content. The followingtable summarizes the results of one such experiment.

TABLE I Sample % Hi-M 36° C., culture medium 19 36° C., culture medium +Betaine 14 37° C., culture medium 18 37° C., culture medium + Betaine 13

Yet further evidence for the correlation between high-mannose contentand osmolality is depicted in FIG. 2. Addition of about 20 mM betaine tocell culture medium dramatically reduced high-mannose content of theexemplary recombinant glycoprotein. For example, when the osmolality wasabout 300 mOsm/Kg, the high mannose content reduced from about 9.5% atabout 0 mM betaine to about 4.5% upon the addition of 20 mM betaine(i.e., about a 5% reduction in high-mannose content). Similarly, whenthe osmolality was about 400 mOsm/Kg, the high mannose content reducedfrom about 16.5% at about 0 mM betaine to about 7.5% upon the additionof 20 mM betaine (i.e., about a 9% reduction in high-mannose content).Further, at osmolality of about 500 mOsm/Kg, the high mannose contentreduced from about 25% at about 0 mM betaine to about 9.5% upon theaddition of about 20 mM betaine (i.e., about 15.5% reduction inhigh-mannose content).

Example 2 Concentration of K+ in the Culture can be Controlled toModulate Mannose-Content of Recombinant Glycoproteins

In a further experiment, the concentration of one or more salts in thecell culture medium was controlled to modulate (e.g., reduce) themannose-content (e.g., high-mannose content) of the exemplaryrecombinant glycoprotein. In an exemplary experiment, the concentrationof K+ in the cell culture medium was controlled and shown to affectmannose-content and specifically, the high-mannose content of theexemplary recombinant glycoprotein. Specifically, high-mannose content(i.e., M5 species or greater) of the exemplary recombinant glycoproteinproduced by culturing a host-cell expressing the glycoprotein at either15 mM or 45 mM was examined.

As shown in FIGS. 3 and 4, the percentage of high-mannose contentincreased from about 3% to about 13% with the concomitant increase inosmolality. An osmolality of between about 370 and about 500 mOsm/Kg ledto an increase in high-mannose content that exceeded 10% of theglycoprotein composition.

In a further experiment, it was demonstrated, as shown in FIG. 5, thatoptimum concentration range for K+ concentration in the cell culturemedium is about 0 mM to about 70 mM in order to keep the percentage ofhigh-mannose content of a recombinant glycoprotein below 10%.

Example 3 Concentration of Na+ in the Cell Culture Medium can beControlled to Modulate Mannose-Content of Recombinant Glycoproteins

In a further experiment, the concentration of Na+ was controlled tomodulate (e.g., reduce) high-mannose content of the exemplaryrecombinant glycoprotein. In an exemplary experiment, an increase in Na+concentration in the cell culture medium was shown to contribute to anincrease in the percentage of the high-mannose content of the exemplaryrecombinant glycoprotein.

FIG. 6 demonstrates that the optimum concentration range for Na+ isbetween about 0 mM and about 200 mM in order to keep the percentage ofthe high-mannose content below 10%.

Example 4 Amino Acids Contribute to High-Mannose Content of RecombinantGlycoproteins

In another experiment, the effect of amino acids present in a cellculture medium was examined on the high-mannose content of the exemplaryrecombinant glycoprotein. As shown in FIG. 7, the percentage ofhigh-mannose content of a recombinant glycoprotein increases from about4% to about 10% by doubling the concentration of 20 amino acids in thefeed medium. This experiment demonstrated that a medium enriched foramino-acids results in an increase in the content of high-mannoseglycoproteins expressed by a host-cell cultured in such medium.

Example 5 Overall Composition of the Feed Medium Composition canContribute to High-Mannose Content of Recombinant Glycoproteins

In this experiment, the effect of different types of feed media wasexamined on the high-mannose content of an exemplary recombinantglycoprotein. Specifically, the effect of a modified feed mediumsubstantially-free of the amino acids L-Alanine, L-Arginine HCL,L-Aspartic Acid and L-Glutamic Acid, and also having a lowerconcentration of CaCl, MgCl, KCl and sodium pyruvate relative tounmodified medium was investigated on the high-mannose content of theexemplary recombinant glycoprotein. As depicted in FIG. 8, thehigh-mannose content was about 4% when the modified feed medium was usedand this percentage increased to about 13% when the unmodified feedmedium was used.

Example 6 Effect of Temperature on High-Mannose Content

The effect of four different temperatures on high-mannose content wasexamined using two different feed media. The following data in Table IIindicates that there was an increase in the percentage of high-mannosecontent with an increase in temperature.

TABLE II 36° C. 35° C. 34° C. Conc. % Hi- Conc. % Hi- Conc. % Hi- SampleName (g/L) Man (g/L) Man (g/L) Man feed medium 1 3.2 14 3.1 17 2.8 22feed medium 2 2.0 13 2.1 13 2.1 14

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments in this disclosureand should not be construed to limit its scope. The skilled artisanreadily recognizes that many other embodiments are encompassed by thisdisclosure. All publications and patents cited and sequences identifiedby accession or database reference numbers in this disclosure areincorporated by reference in their entirety. To the extent the materialincorporated by reference contradicts or is inconsistent with thepresent specification, the present specification will supercede any suchmaterial. The citation of any references herein is not an admission thatsuch references are prior art to the present disclosure.

Unless otherwise indicated, all numbers expressing quantities ofingredients, cell culture, treatment conditions, and so forth used inthe specification, including claims, are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessotherwise indicated to the contrary, the numerical parameters areapproximations and may very depending upon the desired properties soughtto be obtained by the present invention. Unless otherwise indicated, theterm “at least” preceding a series of elements is to be understood torefer to every element in the series. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

1. (canceled)
 2. A method of producing a composition of recombinant antibody, or an antigen-binding fragment thereof, comprising culturing a host-cell which expresses the recombinant antibody or antigen-binding fragment thereof in a culture medium having an osmolality of about 600 mOsm/Kg or less, wherein (i) the culture medium comprises a salt selected from the group consisting of potassium at a concentration of about 70 mM or less, sodium at a concentration of about 200 mM or less, and combinations thereof, (ii) the culture medium is substantially free of one or more ammo acids selected from the group consisting of alanine, arginine, aspartic acid and glutamic acid, (iii) the culture medium comprises an osmo-protectant selected from the group consisting of betaine, glycine, L-threonine, L-proline, and derivatives thereof, and (iv) fewer than about 10% of the recombinant antibody molecules in the composition have more than 4 mannose residues.
 3. (canceled)
 4. The method of claim 2, wherein the osmolality of the culture medium is between about 250 and about 600 mOsm/Kg.
 5. The method of claim 4, wherein the osmolality of the culture median is between about 250 and about 500 mOsm/Kg.
 6. The method of claim 4, wherein the osmolality of the culture medium is between about 250 and about 380 mOsm/Kg.
 7. (canceled)
 8. The method of claim 2, wherein the culture medium comprises a salt selected from the group consisting of (a) potassium at a concentration of about 10 mM to about 50 mM; (b) sodium at a concentration of about 50 mM to about 100 mM; and (c) combinations of (a) and (b).
 9. (canceled)
 10. The method of claim 2, wherein the culture medium comprises one or more vitamins selected from the group consisting of biotin, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinamide, pyridoxal HCl, pyridoxine HCl, riboflavin, thiamine HCl and cyanocobalamin, at a concentration of about 0.00005 g/L to about 0.9 g/L.
 11. The method of claim 2, wherein the culture medium comprises glucose at a concentration of about 1 mM to about 90 mM.
 12. The method of claim 2, wherein the culture medium comprises one or more peptones selected from the group consisting of yeast extract, yeast hydrolysate, soy peptone, soy hydrolysate, wheat peptone and wheat hydrolysate, at a concentration of about 0.5 g/L to about 60 g/L. 13.-14. (canceled)
 15. The method of claim 2, wherein the osmo-protectant is betaine at a concentration of about 1 mM to about 100 mM.
 16. The method of claim 15, wherein the betaine is present at a concentration from about 20 mM to about 30 mM.
 17. The method of claim 2, wherein the host-cell is cultured for a period of about 5 to about 14 days.
 18. The method of claim 2, wherein the host-cell is cultured at a temperature of about 31° C. to about 38° C.
 19. The method of claim 2, wherein the host cell is a mammalian cell.
 20. The method of claim 19, wherein the mammalian host cell is a CHO cell. 21.-42. (canceled) 